Pharmaceutical composition containing VEGF nanobody and its use

Abstract

The present invention provides a pharmaceutical composition containing a VEGF nanobody and its use. Specifically, the present invention provides a pharmaceutical composition containing a VEGF nanobody, a cell-penetrating peptide and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can effectively penetrate the cell membrane, and the cell-penetrating peptide contained therein can significantly promote the cell membrane permeability of the VEGF nanobody. The pharmaceutical composition of the present invention can effectively penetrate cancer cells and corneal cells. With the increase of the concentration of the cell-penetrating peptide, the uptake effect of the VEGF nanobody on the cell is significantly improved, and at high doses, it has only slight toxicity and has very good safety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceuticals, and more specifically to a pharmaceutical composition containing a VEGF nano antibody and a use thereof. Background Art

[0002] Cell-penetrating peptides (CPPs) are a class of short-chain polypeptides that can penetrate the cell membrane. Studies have found that CPPs can bind to various drug molecules and deliver drugs into cells. Many experiments and reports have explained the transmembrane and cell uptake mechanisms of CPPs, but the exact pathways by which CPPs enter cells still require further study. At present, studies have found that artificially set conditions can affect experimental results. Different CPPs have different uptake pathways when entering different cells, and the cargo carried by CPPs will also affect the internalization mechanism of CPPs. It is worth noting that the internalization pathway of CPPs is not fixed, and many CPPs can be internalized in multiple ways. In addition, the internalization mechanism of CPPs will also change with their own concentration, cell tissue, and cargo carried. The most studied transmembrane peptides include: TAT, Penetratin, Protamine, Transpotan, MPG, etc. Penetratin is derived from the Drosophila antenna protein homology domain and is an amphipathic membrane-penetrating peptide. It is used as a potential drug carrier for various purposes because of its good membrane-penetrating effect. The cationic amino acids arginine and lysine in the penetratin sequence are irreplaceable sites for exerting membrane-penetrating activity. If they are replaced or modified, the membrane-penetrating effect will be reduced. The tryptophan, phenylalanine and isoleucine in the sequence are hydrophobic amino acids that can promote the interaction between the peptide and the cell membrane lipids. The literature Sgolastra F, Deronde BM, Sarapas JM, et al. Designing Mimics of Membrane Active Proteins [J]. Accounts of Chemical Research, 2013, 46 (12): 2977-87 discloses the penetratin derivative RQIKIWFQWRRMKWKK, indicating that hydrophobic residues play an important role in cell uptake. Chinese invention patent CN101355970A discloses the penetratin derivative RQIKIWFWNRRMKWKK. Chinese invention patent CN108976288A discloses the sequences of various Penetratin derivatives. Literature shows that the introduction of hydrophobic amino acids into the Penetratin molecule can enhance the cell penetration ability of the Penetratin derivatives.

[0003] VEGF nano antibodies have the characteristics of high stability, good water solubility, simple humanization, high targeting, strong penetrability, etc., and play an unimaginable role in immune experiments, diagnosis and treatment. If VEGF nano antibodies are used in combination with cell-penetrating peptides, the utilization rate of VEGF nano antibody drugs can be improved, and further, it is possible to develop highly effective VEGF nano antibody anti-tumor drug preparations and VEGF nano antibody ophthalmic preparations administered by eye drops. At present, there is no public pharmaceutical composition of VEGF nano antibodies with high efficacy and low toxicity combined with cell-penetrating peptides.

[0004] Therefore, developing a VEGF nanoantibody drug composition to improve the cellular uptake effect of VEGF nanoantibodies has good clinical application prospects and can lay a good foundation for preparing VEGF nanoantibody drug preparations with high efficacy and low toxicity, especially for the development of eye drop drug preparations. Summary of the invention

[0005] The object of the present invention is to provide a pharmaceutical composition containing VEGF nanobody and its use.

[0006] The first aspect of the present invention provides a pharmaceutical composition, which contains:

[0007] (a) Anti-VEGF nanobody;

[0008] (b) a cell-penetrating peptide; and

[0009] (c) a pharmaceutically acceptable carrier.

[0010] In another preferred example, the complementarity determining region (CDR) of the anti-VEGF nanobody is CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3.

[0011] In another preferred example, the amino acid sequence of the anti-VEGF nanobody is as shown in SEQ ID NO:4.

[0012] In another preferred embodiment, the cell-penetrating peptide includes Penetratin and its derivatives.

[0013] In another preferred embodiment, the amino acid sequence of Penetratin is shown in SEQ ID NO:5.

[0014] In another preferred embodiment, the Penetratin derivative is a derivative polypeptide that adds, deletes, modifies and / or replaces at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid on the basis of the amino acid sequence shown in SEQ ID NO:5 and retains the transmembrane activity.

[0015] In another preferred embodiment, the derivative polypeptide retains ≥80%, preferably ≥90%; more preferably ≥95% of the membrane-penetrating activity of the polypeptide shown in SEQ ID NO:5.

[0016] In another preferred embodiment, the identity of the derivative polypeptide to SEQ ID NO: 5 is ≥ 80%, preferably ≥ 90%; more preferably ≥ 95%.

[0017] In another preferred example, the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0018] In another preferred embodiment, the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO:7.

[0019] In another preferred embodiment, the pharmaceutical composition contains:

[0020] (a) an anti-VEGF nanobody with an amino acid sequence as shown in SEQ ID NO: 4;

[0021] (b) a cell-penetrating peptide with an amino acid sequence as shown in SEQ ID NO: 7; and

[0022] (c) a pharmaceutically acceptable carrier.

[0023] In another preferred embodiment, the molar ratio of the anti-VEGF nanobody to the cell-penetrating peptide is 1:20 to 10:1, preferably 1:10 to 2:1, and more preferably 1:5 to 1:1.

[0024] In another preferred embodiment, the molar ratio of the anti-VEGF nanobody to the cell-penetrating peptide is 1:3 to 1:1.

[0025] In another preferred embodiment, the total content of the anti-VEGF nanobody and the cell-penetrating peptide is 1 to 99 wt % of the composition, and more preferably 5 to 90 wt %.

[0026] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a lyophilized preparation and a liquid preparation.

[0027] In another preferred embodiment, the dosage form of the pharmaceutical composition includes injections (such as periocular and intraocular injections), eye drops, eye gels or eye ointments.

[0028] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained-release dosage form or a non-sustained-release dosage form.

[0029] In another preferred embodiment, the pharmaceutical composition may further contain other active pharmaceutical ingredients, preferably, active ingredients for treating cancer or eye diseases.

[0030] The second aspect of the present invention provides a use of the pharmaceutical composition described in the first aspect of the present invention for preparing a drug for treating VEGF-related diseases or conditions.

[0031] In another preferred embodiment, the VEGF-related diseases or conditions include cancer or eye diseases.

[0032] In another preferred embodiment, the cancer includes but is not limited to: breast cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, thyroid cancer, nasopharyngeal cancer, or a combination thereof.

[0033] In another preferred embodiment, the eye disease is an eye disease associated with neovascularization.

[0034] In another preferred embodiment, the eye disease includes but is not limited to: age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma, or a combination thereof.

[0035] The third aspect of the present invention provides a method for preparing the pharmaceutical composition according to the first aspect of the present invention, comprising the steps of:

[0036] (i) respectively providing anti-VEGF nanobody and cell-penetrating peptide,

[0037] (ii) physically mixing the anti-VEGF nanobody with the cell-penetrating peptide to obtain a mixture, and

[0038] (iii) mixing the mixture with a pharmaceutically acceptable carrier under conditions suitable for preparing drugs, thereby preparing the pharmaceutical composition.

[0039] In another preferred embodiment, in step (ii), the anti-VEGF nanobody can be premixed with any pharmaceutically acceptable carrier to obtain a first premix, and then the first premix is ​​physically mixed with the cell-penetrating peptide to obtain the mixture.

[0040] In another preferred embodiment, in step (ii), the cell-penetrating peptide can be premixed with any pharmaceutically acceptable carrier to obtain a second premix, and then the second premix is ​​physically mixed with the anti-VEGF nanobody to obtain the mixture.

[0041] In another preferred embodiment, in step (ii), the anti-VEGF nanobody and the cell-penetrating peptide can be premixed with any pharmaceutically acceptable carrier, respectively, to obtain a first premix and a second premix, and then the first premix and the second premix are physically mixed to obtain the mixture.

[0042] A fourth aspect of the present invention provides a medicine kit, comprising:

[0043] (i) a first container, and the active ingredient (a) anti-VEGF nanobody or a drug containing the active ingredient (a) contained in the first container;

[0044] (ii) a second container, and the active ingredient (b) cell-penetrating peptide, or a drug containing the active ingredient (b) contained in the second container; and

[0045] (iii) instructions for administering the active ingredient (a) and the active ingredient (b) in combination to treat a VEGF-related disease or condition.

[0046] In another preferred embodiment, the medicines in the first container and the second container are a single preparation containing active ingredient (a) and a single preparation containing active ingredient (b).

[0047] A fifth aspect of the present invention provides a method for treating a VEGF-related disease or condition, wherein the method comprises the steps of:

[0048] (i) administering the active ingredient (a) anti-VEGF nanobody and the active ingredient (b) transmembrane peptide to a subject in need thereof, or administering the pharmaceutical composition described in the first aspect of the present invention.

[0049] In another preferred embodiment, the administration includes simultaneous administration or sequential administration.

[0050] In another preferred embodiment, the subject includes humans and non-human mammals.

[0051] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1The results show that the penetrating peptide Penetratin and its mutants combined with VEGF nanoantibodies can effectively penetrate the cell membrane after being mixed with VEGF nanoantibodies, and bring VEGF nanoantibodies into cells. Its penetrating effect is significantly better than that of wild-type Penetratin-WT and mutant Penetratin-Mut 1.

[0053] Figure 2A and Figure 2B The results show that the penetration effect of different VEGF antibodies and penetrating peptide mixtures in different types of cells. The results show that the penetration effect of VEGF nanoantibodies mixed with Penetratin-Mut2 on various cells is significantly better than that of the commercial monoclonal antibody Eylea and Penetratin-Mut2 mixture; and as the concentration of Penetratin-Mut2 increases, the uptake effect of VEGF nanoantibodies on various types of cells is significantly improved.

[0054] Figure 3A and Figure 3B The toxicity of the mixture of VEGF nanobody and penetratin peptide on different types of cells was shown. The results showed that the low-dose group (9μM) VEGF nanobody, Penetratin-Mut2, Penetratin-Mut3, VEGF nanobody / Penetratin-Mut2 mixture (3:1), and VEGF nanobody / Penetratin-Mut3 mixture (3:1) had basically no toxic side effects on the five types of cells; while the high-dose group (90μM) VEGF nanobody / Penetratin-Mut2 mixture showed slight toxicity to colon adenocarcinoma cells HT29 and breast cancer cells MCF7 cells, and the high-dose group (90μM) VEGF nanobody / Penetratin-Mut3 mixture showed obvious toxicity to colon adenocarcinoma cells HT29 and breast cancer cells MCF7 cells. Among them, the dose of the mixture of VEGF nanobody and penetratin peptide (9μM or 90μM) is the content of the nanobody in the mixture. DETAILED DESCRIPTION

[0055] After extensive and in-depth research and a large number of screenings, the inventors unexpectedly discovered for the first time a pharmaceutical composition formed by a simple physical mixture of a VEGF nano antibody and a cell-penetrating peptide. Experiments show that the pharmaceutical composition of the present invention can effectively penetrate the cell membrane, and the cell-penetrating peptide can effectively bring the VEGF nano antibody into the cell; after the VEGF nano antibody of the present invention is mixed with the cell-penetrating peptide, the permeation and permeation effect on a variety of cells is significantly better than that of a mixture of the commercially available monoclonal antibody Eylea (i.e., a conventional VEGF monoclonal antibody) and the cell-penetrating peptide; the VEGF nano antibody pharmaceutical composition of the present invention has no cytotoxicity at low doses and only slight toxicity at high doses, and is very safe.

[0056] Specifically, the present invention provides a VEGF nanoantibody pharmaceutical composition, which can effectively penetrate the cell membrane and significantly promote the cell membrane permeability of the VEGF nanoantibody; the VEGF nanoantibody pharmaceutical composition of the present invention can effectively penetrate cancer cells and corneal cells, and as the concentration of the cell-penetrating peptide increases, the VEGF nanoantibody's cell uptake effect is significantly improved.

[0057] the term

[0058] As used herein, the terms "VEGF nanobody of the present invention", "nanobody of the present invention", "anti-VEGF nanobody", "VEGF nanobody" have the same meaning and are used interchangeably, all referring to nanobodies that specifically recognize and bind to VEGF (including human VEGF). In a preferred embodiment, the amino acid sequence of the VEGF nanobody of the present invention is shown in SEQ ID NO: 4.

[0059] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0060] As used herein, the terms "single domain", "VHH", "nanobody", "heavy chain antibody" (singledomain antibody, sdAb, or nanobody) have the same meaning and are used interchangeably, referring to cloning the variable region of the antibody heavy chain to construct a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0061] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a b-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial b-folded structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0062] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0063] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3, 2 or 1 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0064] Table A

[0065] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0066] Pharmaceutical composition

[0067] The present invention provides a pharmaceutical composition, which contains (a) an anti-VEGF nanobody; (b) a cell-penetrating peptide; and (c) a pharmaceutically acceptable carrier.

[0068] The "active ingredients" in the pharmaceutical composition of the present invention refer to anti-VEGF nanoantibodies and cell-penetrating peptides. The cell-penetrating peptides can effectively bring VEGF nanoantibodies into cells and can be used to treat VEGF-related cancers and eye diseases.

[0069] "Safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. When the pharmaceutical composition of the present invention is used, the total daily dose generally used in most cases should be lower than (or equal to or slightly greater than) the daily dose of each single drug in a few cases. Of course, the effective dose of the active ingredient used may vary depending on the mode of administration and the severity of the disease to be treated.

[0070] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, sugar alcohol, surfactant, powder, pH adjuster, antibacterial agent, preservative, thickener, stabilizer, nutrient and combination thereof.

[0071] The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under aseptic conditions. The pharmaceutical combination of the present invention can also be prepared as a powder for aerosol inhalation.

[0072] In the present invention, the pharmaceutical composition can be administered to the eye by subretinal or intravitreal injection. A preferred dosage form is an injection.

[0073] The pharmaceutical compositions of the present invention may also be used in combination with other therapeutic agents, such as therapeutic agents for treating cancer or eye diseases.

[0074] application

[0075] As described above, the nano-antibody pharmaceutical composition of the present invention has a wide range of biological and clinical application values, and its application involves the diagnosis and treatment of diseases related to VEGF, basic medical research, biological research and other fields. A preferred application is for clinical diagnosis and targeted therapy of VEGF, including VEGF-related cancers and eye diseases.

[0076] The present invention also provides a method for treating VEGF-related diseases or conditions using the pharmaceutical composition of the present invention, comprising administering the active ingredient (a) anti-VEGF nanoantibody and the active ingredient (b) transmembrane peptide to a subject in need, or administering the pharmaceutical composition described in the first aspect of the present invention.

[0077] When the two active ingredients of the present invention are used for the above purposes, they can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspending agents), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agents in isotonic media). For example, these pharmaceutical preparations can contain about 0.01-99%, more preferably about 0.1%-90% (by weight) of the active ingredient mixed with a carrier.

[0078] The two active ingredients or pharmaceutical compositions of the present invention can be administered by conventional routes, including (but not limited to): intraocular, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration. Preferred routes of administration include oral administration, intraocular administration or intravenous administration.

[0079] The main advantages of the present invention include:

[0080] (a) The VEGF nanobody pharmaceutical composition of the present invention can effectively penetrate the cell membrane and can significantly promote the cell membrane penetration of the VEGF nanobody.

[0081] (b) The VEGF nanobody pharmaceutical composition of the present invention can effectively penetrate cancer cells and corneal cells, and as the concentration of the cell-penetrating peptide increases, the VEGF nanobody's uptake effect on the cells is significantly improved.

[0082] (c) The VEGF nanobody pharmaceutical composition of the present invention has no cytotoxicity at low doses and only slight toxicity at high doses.

[0083] The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0084] general material

[0085] The membrane-penetrating peptide Penetratin used in the examples was prepared using the reference "Sgolastra F, Deronde BM, Sarapas JM, et al. Designing Mimics of Membrane Active Proteins [J]. Accounts of Chemical Research, 2013, 46 (12): 2977-87", and its mutants were prepared using the references "Chinese Invention Patent CN101355970A", "Sgolastra F, Deronde BM, Sarapas JM, et al. Designing Mimics of Membrane Active Proteins [J]. Accounts of Chemical Research, 2013, 46 (12): 2977-87", and "Chinese Invention Patent CN108976288A".

[0086] VEGF nanobody is a nanobody that binds to VEGF independently developed by the applicant, and its amino acid sequence is shown in SEQ ID NO: 4. The monoclonal antibody Eylea used as a control in the examples is a conventional monoclonal antibody that binds to VEGF in the art, and was purchased from Bayer (China) Co., Ltd.

[0087] Example 1 Penetrating effect of penetratin and its mutants combined with VEGF nanobody

[0088] The malignant melanoma A375 cells were digested, neutralized with complete medium, centrifuged and the supernatant was removed, the cells were resuspended with fresh medium, inoculated into 6-well plates, 5E5 cells per well, and cultured for 24 hours; three penetrating peptides Penetratin-WT (amino acid sequence as shown in SEQ ID NO: 5), Penetratin-Mut1 (amino acid sequence as shown in SEQ ID NO: 6), Penetratin-Mut2 (amino acid sequence as shown in SEQ ID NO: 7) were mixed with VEGF nanobody (amino acid sequence as shown in SEQ ID NO: 4) in advance and left at room temperature for 1 day; the cell culture supernatant was discarded on the second day, the DMEM blank medium was washed twice, and then DMEM medium containing the penetrating peptide / antibody mixture was added (the added DMEM medium contained 9uM nanobody, and the molar ratio of each penetrating peptide to antibody was 3: 1), and the culture was continued for 4 hours; and then 10mM containing 0.02mg / ml heparin sodium was used The cells were washed twice with PBS, the supernatant was discarded and trypsin was added to digest the cells, the cells were collected and transferred to a 96-well plate, and the fixative was added and placed at room temperature for 20 minutes; the cells were then washed with PBS and incubated with methanol for 10 minutes; the cells were washed twice with PBS, diluted biotinylated goat anti-VEGF polyclonal antibody (used at a dilution of 1:500) was added, and incubated at room temperature for 20 minutes; the cells were washed once with PBS, and diluted SA-PE fluorescent antibody was added; after the cells were washed with PBS, the PE signal was detected by flow cytometry.

[0089] The results are as follows Figure 1 As shown, after Penetratin-Mut2 is mixed with VEGF nanoantibody, it can effectively penetrate the cell membrane and bring VEGF nanoantibody into the cell. Its membrane penetration effect is significantly better than that of wild-type Penetratin-WT and mutant Penetratin-Mut 1.

[0090] Example 2 Penetration effects of different VEGF antibody and cell-penetrating peptide mixtures in different cell types

[0091] Five types of cells, namely, malignant melanoma cells A375, colon adenocarcinoma cells HT29, lung cancer cells H1299, breast cancer cells MCF7, and corneal epithelial cells HCEC, were digested, neutralized with complete medium, centrifuged and the supernatant removed, the cells were resuspended with fresh medium, inoculated into a 6-well plate, 5E5 / mL cells per well, and cultured for 24 hours; Penetratin-Mut2 was mixed with VEGF nanoantibodies and commercially available monoclonal antibodies Eylea in different molar ratios in advance and placed at room temperature for 1 day; the cell culture supernatant was discarded the next day, the cells were washed twice with DMEM blank medium, and then DMEM medium containing a membrane-penetrating peptide / antibody mixture was added (the added DMEM medium contained 9uM antibody, and the molar ratio of membrane-penetrating peptide to antibody was 1:1, 2:1 or 3:1), and the culture was continued for 4 hours; then 10mM heparin containing 0.02mg / ml was used. The cells were washed twice with PBS, the supernatant was discarded and trypsin was added to digest the cells, the cells were collected and transferred to a 96-well plate, and the fixative was added and placed at room temperature for 20 minutes; the cells were then washed with PBS and incubated with methanol for 10 minutes; the cells were washed twice with PBS, and for the VEGF nanoantibody group, diluted biotinylated goat anti-VEGF polyclonal antibody (used at a dilution of 1:500) was added, incubated at room temperature for 20 minutes, the cells were washed once with PBS, and diluted SA-PE fluorescent antibody was added; for the commercially available monoclonal antibody Eylea group, a 1:200 diluted Goat anti human IgG-PE antibody was added; after washing the cells with PBS, the PE signal was detected by flow cytometry.

[0092] The results are as follows Figure 2A and Figure 2B As shown, the permeation and transmembrane effect of VEGF nanobody mixed with Penetratin-Mut2 on various cells is significantly better than that of the commercially available monoclonal antibody Eylea and Penetratin-Mut2 mixture; and as the concentration of Penetratin-Mut2 increases, the uptake effect of VEGF nanobody on various types of cells is significantly improved.

[0093] Example 3 Toxicity of a mixture of VEGF nanobody and cell-penetrating peptide to different cell types

[0094] Five cell types, namely, malignant melanoma cells A375, colon adenocarcinoma cells HT29, lung cancer cells H1299, breast cancer cells MCF7, and corneal epithelial cells HCEC, were digested, neutralized with complete medium, centrifuged, and the supernatant was removed. The cells were resuspended in fresh medium and plated in a 96-well plate at 2E4 / well, and cultured at 37°C, 5% CO2 for 24 h. The supernatant was discarded, and 100 uL / well of VEGF nanoantibodies, Penetratin-Mut2, and Penetratin-Mut3 (amino acid sequence as shown in SEQ ID NO:8), Penetratin-Mut2 / VEGF nanoantibody mixture (3:1), Penetratin-Mut3 / VEGF nanoantibody mixture (3:1) (the order of VEGF nanoantibody and membrane-penetrating peptide was adjusted according to the data shown in Figure 3, please review) DMEM culture medium, continue to culture for 4 hours, 37°C, 5% CO2; discard the supernatant, wash the cells once with complete culture medium, add new complete culture medium 100uL / well, and continue to culture for 24 hours; add CCK8 10uL to each well, incubate at 37°C for 4 hours, and read OD450 with a microplate reader.

[0095] The results are as follows Figure 3A and Figure 3B As shown, the low-dose group (9 μM) VEGF nanoantibody, Penetratin-Mut2, Penetratin-Mut3, Penetratin-Mut2 / VEGF nanoantibody mixture (3:1), and Penetratin-Mut3 / VEGF nanoantibody mixture (3:1) had basically no toxic side effects on the five types of cells; while the high-dose group (90 μM) VEGF nanoantibody / Penetratin-Mut2 mixture showed slight toxic effects on the five types of cells, and the high-dose group (90 μM) VEGF nanoantibody / Penetratin-Mut3 mixture showed obvious toxic effects on the five types of cells.

[0096] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Shanghai Luoqi Biopharmaceutical Technology Co., Ltd. <120> Pharmaceutical composition containing VEGF nanobody and its use <130> P2020-2429 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Gly Phe Thr Phe Asp Asp Pro Asp Val Gly 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <400> 2 Ile Ser Lys Asp Gly Ser Thr 1 5 <210> 3 <211> 19 <212> PRT <213> Artificial Sequence <400> 3 Ala Ala Asp Ser Asn Pro Ile Ala Pro Ile Arg Thr Cys Leu Gly Trp 1 5 10 15 Tyr Asn Tyr <210> 4 <211> 259 <212> PRT <213> Artificial Sequence <400> 4 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Asp Pro 20 25 30 Asp Val Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val 35 40 45 Ser Thr Ile Ser Lys Asp Gly Ser Thr Tyr Tyr Thr Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Tyr Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Asp Ser Asn Pro Ile Ala Pro Ile Arg Thr Cys Leu Gly Trp Tyr 100 105 110 Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Gln Glu Ser Gly Gly Gly 130 135 140 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly 145 150 155 160 Phe Thr Phe Asp Asp Pro Asp Val Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Asn Glu Cys Glu Leu Val Ser Thr Ile Ser Lys Asp Gly Ser Thr Tyr 180 185 190 Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Tyr Ala 195 200 205 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Ala Ala Asp Ser Asn Pro Ile Ala Pro Ile Arg 225 230 235 240 Thr Cys Leu Gly Trp Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr 245 250 255 Val Ser Ser <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <400> 5 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <400> 6 Arg Gln Ile Lys Ile Trp Phe Trp Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <400> 7 Arg Gln Ile Lys Ile Trp Phe Gln Trp Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 8 Arg Trp Ile Lys Ile Trp Phe Trp Trp Arg Arg Met Lys Trp Lys Lys 1 5 10 15

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition is composed of the following ingredients: (a) an anti-VEGF nanobody with an amino acid sequence as shown in SEQ ID NO: 4; (b) a cell-penetrating peptide with an amino acid sequence as shown in SEQ ID NO: 7 or 8; and (c) a pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of the anti-VEGF nanobody to the cell-penetrating peptide is 1:20 to 10:

1.

3. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of the anti-VEGF nano antibody to the cell-penetrating peptide is 1:10 to 2:

1.

4. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of the anti-VEGF nanobody to the cell-penetrating peptide is 1:5 to 1:

1.

5. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of the anti-VEGF nano antibody to the cell-penetrating peptide is 1:3 to 1:

1.

6. The pharmaceutical composition according to claim 1, characterized in that The dosage forms of the pharmaceutical composition include injection, eye drops, eye gel or eye ointment.

7. Use of the pharmaceutical composition according to claim 1, characterized in that: For preparing a drug for treating VEGF-related cancers and eye diseases, wherein the cancer is selected from the group consisting of melanoma, breast cancer, lung cancer, colon adenocarcinoma, or a combination thereof; The eye disease is an eye disease associated with neovascularization, and the eye disease is selected from the following group: age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma, or a combination thereof.

8. A method for preparing the pharmaceutical composition according to claim 1, characterized in that: Includes steps: (i) respectively providing an anti-VEGF nanobody and a cell-penetrating peptide, wherein the amino acid sequence of the anti-VEGF nanobody is as shown in SEQ ID NO: 4; the amino acid sequence of the cell-penetrating peptide is as shown in SEQ ID NO: 7 or SEQ ID NO: 8, (ii) physically mixing the anti-VEGF nanobody with the cell-penetrating peptide to obtain a mixture, and (iii) mixing the mixture with a pharmaceutically acceptable carrier under conditions suitable for preparing drugs, thereby preparing the pharmaceutical composition.

9. A medicine box, characterized in that: The kit contains: (i) a first container, and an active ingredient (a) anti-VEGF nanobody contained in the first container, or a drug containing the active ingredient (a), wherein the amino acid sequence of the anti-VEGF nanobody is as shown in SEQ ID NO: 4; (ii) a second container, and an active ingredient (b) a cell-penetrating peptide contained in the second container, or a drug containing the active ingredient (b), wherein the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO: 7 or SEQ ID NO: 8; and (iii) instructions for administering the active ingredient (a) and the active ingredient (b) in combination to treat a VEGF-related disease or condition.

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